Proteolysis of Virulence Regulator Toxr is Associated with Entry of Vibrio Cholerae Into a Dormant State

Vibrio cholerae O1 is a natural inhabitant of aquatic environments and causes the diarrheal disease, cholera. Two of its primary virulence regulators, TcpP and ToxR, are localized in the inner membrane. TcpP is encoded on the Vibrio Pathogenicity Island (VPI), a horizontally acquired mobile genetic element, and functions primarily in virulence gene regulation. TcpP has been shown to undergo regulated intramembrane proteolysis (RIP) in response to environmental conditions that are unfavorable for virulence gene expression. ToxR is encoded in the ancestral genome and is present in non-pathogenic strains of V. cholerae, indicating it has roles outside of the human host. In this study, we show that ToxR undergoes RIP in V. cholerae in response to nutrient limitation at alkaline pH, a condition that occurs during the stationary phase of growth. This process involves the site-2 protease RseP (YaeL), and is dependent upon the RpoE-mediated periplasmic stress response, as deletion mutants for the genes encoding these two proteins cannot proteolyze ToxR under nutrient limitation at alkaline pH. We determined that the loss of ToxR, genetically or by proteolysis, is associated with entry of V. cholerae into a dormant state in which the bacterium is normally found in the aquatic environment called viable but nonculturable (VBNC). Strains that can proteolyze ToxR, or do not encode it, lose culturability, experience a change in morphology associated with cells in VBNC, yet remain viable under nutrient limitation at alkaline pH. On the other hand, mutant strains that cannot proteolyze ToxR remain culturable and maintain the morphology of cells in an active state of growth. Overall, our findings provide a link between the proteolysis of a virulence regulator and the entry of a pathogen into an environmentally persistent state

The Fatty Acid Regulator FadR Influences the Expression of the Virulence Cascade in the El Tor Biotype of Vibrio cholerae by Modulating the Levels of ToxT via Two Different Mechanisms

FadR is a master regulator of fatty acid (FA) metabolism that coordinates the pathways of FA degradation and biosynthesis in enteric bacteria. We show here that a ΔfadR mutation in the El Tor biotype of Vibrio cholerae prevents the expression of the virulence cascade by influencing both the transcription and the posttranslational regulation of the master virulence regulator ToxT. FadR is a transcriptional regulator that represses the expression of genes involved in FA degradation, activates the expression of genes involved in unsaturated FA (UFA) biosynthesis, and also activates the expression of two operons involved in saturated FA (SFA) biosynthesis. Since FadR does not bind directly to the toxT promoter, we determined whether the regulation of any of its target genes indirectly influenced ToxT. This was accomplished by individually inserting a double point mutation into the FadR-binding site in the promoter of each target gene, thereby preventing their activation or repression. Although preventing FadR-mediated activation of fabA, which encodes the enzyme that carries out the first step in UFA biosynthesis, did not significantly influence either the transcription or the translation of ToxT, it reduced its levels and prevented virulence gene expression. In the mutant strain unable to carry out FadR-mediated activation of fabA, expressing fabA ectopically restored the levels of ToxT and virulence gene expression. Taken together, the results presented here indicate that V. cholerae FadR influences the virulence cascade in the El Tor biotype by modulating the levels of ToxT via two different mechanisms

The 40-Residue Insertion in Vibrio Cholerae FadR Facilitates Binding of an Additional Fatty acyl-CoA Ligand

FadR is a master regulator of fatty acid metabolism and influences virulence in certain members of Vibrionaceae. Among FadR homologues of the GntR family, the Vibrionaceae protein is unusual in that it contains a C-terminal 40-residue insertion. Here we report the structure of Vibrio cholerae FadR (VcFadR) alone, bound to DNA, and in the presence of a ligand, oleoyl-CoA. Whereas Escherichia coli FadR (EcFadR) contains only one acyl-CoA-binding site in each monomer, crystallographic and calorimetric data indicate that VcFadR has two. One of the binding sites resembles that of EcFadR, whereas the other, comprised residues from the insertion, has not previously been observed. Upon ligand binding, VcFadR undergoes a dramatic conformational change that would more fully disrupt DNA binding than EcFadR. These findings suggest that the ability to bind and respond to an additional ligand allows FadR from Vibrionaceae to function as a more efficient regulator

Introduction: Examined Live – An Epistemological Exchange Between Philosophy and Cultural Psychology on Reflection

Besides the general agreement about the human capability of reflection, there is a large area of disagreement and debate about the nature and value of “reflective scrutiny” and the role of “second-order states” in everyday life. This problem has been discussed in a vast and heterogeneous literature about topics such as epistemic injustice, epistemic norms, agency, understanding, meta-cognition etc. However, there is not yet any extensive and interdisciplinary work, specifically focused on the topic of the epistemic value of reflection. This volume is one of the first attempts aimed at providing an innovative contribution, an exchange between philosophy, epistemology and psychology about the place and value of reflection in everyday life. Our goal in the next sections is not to offer an exhaustive overview of recent work on epistemic reflection, nor to mimic all of the contributions made by the chapters in this volume. We will try to highlight some topics that have motivated a new resumption of this field and, with that, drawing on chapters from this volume where relevant. Two elements defined the scope and content of this volume, on the one hand, the crucial contribution of Ernest Sosa, whose works provide original and thought-provoking contributions to contemporary epistemology in setting a new direction for old dilemmas about the nature and value of knowledge, giving a central place to reflection. On the other hand, the recent developments of cultural psychology, in the version of the “Aalborg approach”, reconsider the object and scope of psychological sciences, stressing that “[h]uman conduct is purposeful”

Bird pollination of Canary Island endemic plants

The Canary Islands are home to a guild of endemic, threatened bird pollinated plants. Previous work has suggested that these plants evolved floral traits as adaptations to pollination by flower specialist sunbirds, but subsequently they appear to be have co-opted passerine birds as sub-optimal pollinators. To test this idea we carried out a quantitative study of the pollination biology of three of the bird pollinated plants, Canarina canariensis (Campanulaceae), Isoplexis canariensis (Veronicaceae) and Lotus berthelotii (Fabaceae), on the island of Tenerife. Using colour vision models, we predicted the detectability of flowers to bird and bee pollinators. We measured pollinator visitation rates, nectar standing crops, as well as seed set and pollen removal and deposition. These data showed that the plants are effectively pollinated by non-flower specialist passerine birds that only occasionally visit flowers. The large nectar standing crops and extended flower longevities (>10days) of Canarina and Isoplexis suggests that they have evolved bird pollination system that effectively exploits these low frequency non-specialist pollen vectors and is in no way suboptimal. Seed set in two of the three species was high, and was significantly reduced or zero in flowers where pollinator access was restricted. In L. berthelotii, however, no fruit set was observed, probably because the plants were self incompatible horticultural clones of a single genet. We also show that, while all three species are easily detectable for birds, the orange Canarina and the red Lotus (but less so the yellow-orange Isoplexis) should be difficult to detect for insect pollinators without specialised red receptors, such as bumblebees. Contrary to expectations if we accept that the flowers are primarily adapted to sunbird pollination, the chiffchaff (Phylloscopus canariensis) was an effective pollinator of these species

Facts, Principles, and (Real) Politics

Should our factual understanding of the world influence our normative theorising about it? G.A. Cohen has argued that our ultimate normative principles should not be constrained by facts. Many others have defended or are committed to various versions or subsets of that claim. In this paper I dispute those positions by arguing that, in order to resist the conclusion that ultimate normative principles rest on facts about possibility or conceivability, one has to embrace an unsatisfactory account of how principles generate normative political judgments. So political theorists have to choose between principles ostensibly unbiased by our current understanding of human motivation and political reality, or principles capable of reliably generating political judgments. I conclude with wider methodological observations in defence of the latter option, and so of a return to political philosophy’s traditional blend of normative and descriptive elements

The genomes of two key bumblebee species with primitive eusocial organization

Background: The shift from solitary to social behavior is one of the major evolutionary transitions. Primitively eusocial bumblebees are uniquely placed to illuminate the evolution of highly eusocial insect societies. Bumblebees are also invaluable natural and agricultural pollinators, and there is widespread concern over recent population declines in some species. High-quality genomic data will inform key aspects of bumblebee biology, including susceptibility to implicated population viability threats. Results: We report the high quality draft genome sequences of Bombus terrestris and Bombus impatiens, two ecologically dominant bumblebees and widely utilized study species. Comparing these new genomes to those of the highly eusocial honeybee Apis mellifera and other Hymenoptera, we identify deeply conserved similarities, as well as novelties key to the biology of these organisms. Some honeybee genome features thought to underpin advanced eusociality are also present in bumblebees, indicating an earlier evolution in the bee lineage. Xenobiotic detoxification and immune genes are similarly depauperate in bumblebees and honeybees, and multiple categories of genes linked to social organization, including development and behavior, show high conservation. Key differences identified include a bias in bumblebee chemoreception towards gustation from olfaction, and striking differences in microRNAs, potentially responsible for gene regulation underlying social and other traits. Conclusions: These two bumblebee genomes provide a foundation for post-genomic research on these key pollinators and insect societies. Overall, gene repertoires suggest that the route to advanced eusociality in bees was mediated by many small changes in many genes and processes, and not by notable expansion or depauperation

Chromatic Signals Control Proboscis Movements during Hovering Flight in the Hummingbird Hawkmoth Macroglossum stellatarum

Most visual systems are more sensitive to luminance than to colour signals. Animals resolve finer spatial detail and temporal changes through achromatic signals than through chromatic ones. Probably, this explains that detection of small, distant, or moving objects is typically mediated through achromatic signals. Macroglossum stellatarum are fast flying nectarivorous hawkmoths that inspect flowers with their long proboscis while hovering. They can visually control this behaviour using floral markings known as nectar guides. Here, we investigate whether this is mediated by chromatic or achromatic cues. We evaluated proboscis placement, foraging efficiency, and inspection learning of naïve moths foraging on flower models with coloured markings that offered either chromatic, achromatic or both contrasts. Hummingbird hawkmoths could use either achromatic or chromatic signals to inspect models while hovering. We identified three, apparently independent, components controlling proboscis placement: After initial contact, 1) moths directed their probing towards the yellow colour irrespectively of luminance signals, suggesting a dominant role of chromatic signals; and 2) moths tended to probe mainly on the brighter areas of models that offered only achromatic signals. 3) During the establishment of the first contact, naïve moths showed a tendency to direct their proboscis towards the small floral marks independent of their colour or luminance. Moths learned to find nectar faster, but their foraging efficiency depended on the flower model they foraged on. Our results imply that M. stellatarum can perceive small patterns through colour vision. We discuss how the different informational contents of chromatic and luminance signals can be significant for the control of flower inspection, and visually guided behaviours in general

Quorum Sensing Regulation of the Two hcp Alleles in Vibrio cholerae O1 Strains

BACKGROUND: The type VI secretion system (T6SS) has emerged as a protein secretion system important to several gram-negative bacterial species. One of the common components of the system is Hcp, initially described as a hemolysin co-regulated protein in a serotype O17 strain of Vibrio cholerae. Homologs to V. cholerae hcp genes have been found in all characterized type VI secretion systems and they are present also in the serotype O1 strains of V. cholerae that are the cause of cholera diseases but seemed to have non-functional T6SS. METHODOLOGY/PRINCIPAL FINDINGS: The serotype O1 V. cholerae strain A1552 was shown to express detectable levels of Hcp as determined by immunoblot analyses using polyclonal anti-Hcp antiserum. We found that the expression of Hcp was growth phase dependent. The levels of Hcp in quorum sensing deficient mutants of V. cholerae were compared with the levels in wild type V. cholerae O1 strain A1552. The expression of Hcp was positively and negatively regulated by the quorum sensing regulators HapR and LuxO, respectively. In addition, we observed that expression of Hcp was dependent on the cAMP-CRP global transcriptional regulatory complex and required the RpoN sigma factor. CONCLUSION/SIGNIFICANCE: Our results show that serotype O1 strains of V. cholerae do express Hcp which is regarded as one of the important T6SS components and is one of the secreted substrates in non-O1 non-O139 V. cholerae isolates. We found that expression of Hcp was strictly regulated by the quorum sensing system in the V. cholerae O1 strain. In addition, the expression of Hcp required the alternative sigma factor RpoN and the cAMP-CRP global regulatory complex. Interestingly, the environmental isolates of V. cholerae O1 strains that showed higher levels of the HapR quorum sensing regulator in comparison with our laboratory standard serotype O1 strain A1552 where also expressing higher levels of Hcp

Strategies of the honeybee Apis mellifera during visual search for vertical targets presented at various heights: a role for spatial attention?

When honeybees are presented with a colour discrimination task, they tend to choose swiftly and accurately when objects are presented in the ventral part of their frontal visual field. In contrast, poor performance is observed when objects appear in the dorsal part. Here we investigate if this asymmetry is caused by fixed search patterns or if bees can use alternative search mechanisms such as spatial attention, which allows flexible focusing on different areas of the visual field. We asked individual honeybees to choose an orange rewarded target among blue distractors. Target and distractors were presented in the ventral visual field, the dorsal field or both. Bees presented with targets in the ventral visual field consistently had the highest search efficiency, with rapid decisions, high accuracy and direct flight paths. In contrast, search performance for dorsally located targets was inaccurate and slow at the beginning of the test phase, but bees increased their search performance significantly after a few learning trials: they found the target faster, made fewer errors and flew in a straight line towards the target. However, bees needed thrice as long to improve the search for a dorsally located target when the target's position changed randomly between the ventral and the dorsal visual field. We propose that honeybees form expectations of the location of the target's appearance and adapt their search strategy accordingly. Different possible mechanisms of this behavioural adaptation are discussed.L.M. was recipient of a DOC-fFORTE fellowship of the Austrian Academy of Science at the Department of Integrative Zoology, University of Vienna. L.C. is supported by an ERC Advanced Grant and a Royal Society Wolfson Research Merit Award